Induced osteogenic differentiation of human smooth muscle cells as a model of vascular calcification

Abstract

Vascular calcification is a severe pathological event in the manifestation of atherosclerosis. Pathogenic triggers mediating osteogenic differentiation of arterial smooth muscle cells (SMC) in humans remain insufficiently understood and are to a large extent investigated in animal models or cells derived thereof. Here, we describe an in vitro model based on SMC derived from healthy and diseased humans that allows to comprehensively investigate vascular calcification mechanisms. Comparing the impact of the commonly used SMC culture media VascuLife, DMEM, and M199, cells were characterised by immunofluorescence, flow cytometry, qPCR, and regarding their contractility and proliferative capacity. Irrespective of the arterial origin, the clinical background and the expansion medium used, all cells expressed typical molecular SMC marker while contractility varied between donors. Interestingly, the ability to induce an osteogenic differentiation strongly depended on the culture medium, with only SMC cultured in DMEM depositing calcified matrix upon osteogenic stimulation, which correlated with increased alkaline phosphatase activity, increased inorganic phosphate level and upregulation of osteogenic gene markers. Our optimized model is suitable for donor-oriented as well as broader screening of potential pathogenic mediators triggering vascular calcification. Translational studies aiming to identify and to evaluate therapeutic targets in a personalized fashion would be feasible.

Figure 1

Donor characteristics, sample overview, and immunofluorescence characterisation of isolated SMC. Cells isolated from various pathologically altered, clinical samples of different arterial origin were analysed morphologically and by immunofluorescence staining of specific marker proteins to confirm their SMC characteristics at passage three. Nuclei (blue) were visualized with DAPI. SMA (red) - smooth muscle actin, and MYH11 (green) - myosin heavy chain 11 were labelled with the respective antibodies.

Figure 2

SMC marker expression after expansion in different media. (a) Histograms of flow cytometric surface marker expression (grey) of CD13, CD14, CD31, CD44, CD45, CD73, CD90, and CD140b on single live cells at passage three or four identified according to the gating strategy shown in Supplementary Fig. S2. Marker expression is given in comparison to the unstained control (white). (b) Gene expression level of SMC marker in isolated cells at passage one or two, normalized to the expression of the housekeeping gene RPL13A. Values of the individual donors are shown as points superimposed onto the boxplots. Expansion medium of each donor is given in brackets; D - DMEM, M - M199, S - supplements (FGF, EGF, insulin, ascorbic acid), VL - VascuLife.

Figure 3

Contractility of SMC varies between donors. SMC expanded in different media were functionally assessed by stimulation with 10 µM carbachol for 15 min in (a) their respective expansion medium (given in brackets) or (b) after subcultivation in VascuLife medium for one passage. For visualization of stimulation-induced cell area change the membrane of single cells was marked; D - DMEM, M - M199, S - supplements (FGF, EGF, insulin, ascorbic acid), VL - VascuLife.

Figure 4

Proliferation of SMC varies between expansion media. SMC of passage three were seeded at 2 × 10³ cells/well and expanded in different media. Cell proliferation was determined at the indicated time points using the CyQuant Proliferation assay and is shown as the fold change to day zero; D - DMEM, M - M199, S - supplements (FGF, EGF, insulin, ascorbic acid), VL - VascuLife.

Figure 5

Osteogenic stimulation elevates alkaline phosphatase activity and phosphate level indicating an osteogenic differentiation of SMC. (a) Alkaline phosphatase (ALP) activity after osteogenic stimulation of SMC in VascuLife, DMEM, or M199. ALP activity was normalized to the metabolic activity of the cells determined with PrestoBlue Cell Viability Reagent. (b) Phosphate (PO4) level determined in the supernatant of SMC osteogenically stimulated in VascuLife, DMEM, or M199. ALP and PO4 values are given as mean of five replicate wells per donor and medium ± standard deviation; D - DMEM, M - M199, S - supplements (FGF, EGF, insulin, ascorbic acid), VL - VascuLife.

Figure 6

Increased gene expression of osteogenic markers after stimulation in VascuLife and DMEM substantiates an osteogenic differentiation of SMC while SMC characteristics are partially maintained. Gene expression analysis of SMC osteogenically stimulated in VascuLife or DMEM for ten days; (a) osteogenic marker, (b) negative regulators of calcification, (c) SMC marker and transcriptional regulators. Expression was normalized to the expression of the housekeeping gene RPL13A and the fold change between day zero (baseline) and day ten was calculated. Expansion medium controls for DMEM and DMEM plus supplements were pooled for quantitative analysis. Gene expression analysis of ID2-derived SMC was not possible due to low cell numbers and resulting insufficient amounts of isolated mRNA. For statistical analysis a Kruskal-Wallis-test with an uncorrected Dunn’s post test was used. Only significant differences (p < 0.05) are given; D - DMEM, M - M199, S - supplements (FGF, EGF, insulin, ascorbic acid), VL - VascuLife, EM - expansion medium, OM - osteogenic medium.

Figure 7

SMC deposit calcified matrix only after osteogenic stimulation in DMEM. Alizarin red staining of calcified matrix deposited by SMC osteogenically stimulated in different culture media for 14 and 21 days. (a) Macroscopic examination of calcified matrix deposition by SMC after 21 days of osteogenic stimulation in VascuLife or DMEM. (b) Microscopic examination of the wells and their respective expansion media controls after 21 days of osteogenic stimulation in VascuLife or DMEM. (c) Quantification of the deposited calcified matrix normalized to the cell number on day 14 and day 21. Expansion medium controls for DMEM and DMEM plus supplements were pooled for quantitative analysis. Values of the individual donors are shown as points superimposed onto the boxplots and are given as mean of five replicate wells per donor and medium. For statistical analysis data of each point in time were compared amongst each other using a Kruskal-Wallis-test with a Dunn’s post test corrected for multiple comparisons. Only significant differences (p < 0.05) are given; D - DMEM, S - supplements (FGF, EGF, insulin, ascorbic acid), VL - VascuLife, EM - expansion medium, OM - osteogenic medium.

Figure 8

Schematic work flow for the in vitro analysis of vascular calcification.